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Hemolymph ionic regulation and adjustments in gill (Na(+), K(+))-ATPase activity during salinity acclimation in the swimming crab Callinectes ornatus (Decapoda, Brachyura)

GARCON, Daniela P.; MASUI, Douglas C.; MANTELATTO, Fernando L. M.; FURRIEL, Rosa P. M.; MCNAMARA, John C.; LEONE, Francisco A.
Fonte: ELSEVIER SCIENCE INC Publicador: ELSEVIER SCIENCE INC
Tipo: Artigo de Revista Científica
ENG
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We evaluate hemolymph osmotic and ionic regulatory abilities and characterize a posterior gill microsomal (Na(+), K(+))-ATPase from the marine swimming crab, Callinectes ornatus, acclimated to 21 parts per thousand or 33 parts per thousand salinity. C ornatus is isosmotic after acclimation to 21 parts per thousand but is hyposmotic at 33 parts per thousand salinity; hemolymph ions do not recover initial levels on acclimation to 21 parts per thousand salinity but are anisoionic compared to ambient concentrations, revealing modest regulatory ability. NH(4)(+) modulates enzyme affinity for K(+), which increases 187-fold in crabs acclimated to 33%. salinity. The (Na(+), K(+))-ATPase redistributes into membrane fractions of different densities, suggesting that altered membrane composition results from salinity acclimation. ATP was hydrolyzed at maximum rates of 182.6 +/- 7.1 nmol Pi min(-1) mg(-1) (21 parts per thousand) and 76.2 +/- 3.5 nmol Pi min(-1) mg(-1) (33 parts per thousand), with little change in K(M) values (approximate to 50 mu mol L(-1)). K(+) together with NH(4)(+) synergistically stimulated activity to maximum rates of approximate to 240 nmol Pi min(-1) mg(-1). K, values for ouabain inhibition (approximate to 110 mu mol L(-1)) decreased to 44.9 +/- 1.0 mu mol L(-1) (21 parts per thousand) and 28.8 +/- 1.3 mu mol L(-1) (33 parts per thousand) in the presence of both K(+) and NH(4)(+). Assays employing various inhibitors suggest the presence of mitochondrial F(0)F(1)- and K(+)- and V-ATPase activities in the gill microsomes. (C) 2009 Elsevier Inc. All rights reserved.; Funda go de Amparo A Pesquisa do Estado de Sao Paulo (FAPESP)...

Effects of Plasmodium gallinaceum on hemolymph physiology of Aedes aegypti during parasite development

ARAUJO, Ricardo Vieira; MACIEL, Ceres; HARTFELDER, Klaus; CAPURRO, Margareth Lara
Fonte: PERGAMON-ELSEVIER SCIENCE LTD Publicador: PERGAMON-ELSEVIER SCIENCE LTD
Tipo: Artigo de Revista Científica
ENG
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Insect disease vectors show diminished fecundity when infected with Plasmodium. This phenomenon has already been demonstrated in laboratory models such as Aedes aegypti, Anopheles gambiae and Anopheles stephensi. This study demonstrates several changes in physiological processes of A. aegypti occurring upon infection with Plasmodium gallinaceum, such as reduced ecdysteroid levels in hemolymph as well as altered expression patterns for genes involved in vitellogenesis, lipid transport and immune response. Furthermore, we could show that P. gallinaceum infected A. aegypti presented a reduction in reproductive fitness, accompanied by an activated innate immune response and increase in lipophorin expression, with the latter possibly representing a nutritional resource for Plasmodium sporozoites. (C) 2010 Elsevier Ltd. All rights reserved.; Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP); Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP); Ministério da Saúde MTC/DECIT; Ministério da Saúde MTC/DECIT; Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)

Estudo do efeito de uma proteína antiapoptótica obtida da hemolinfa de Lonomia obliqua sobre as mitocôndrias de células Sf-9.; Effect of an anti-apoptotic protein obtained from the hemolymph of Lonomia obliqua on the mitochondria of cells Sf-9.

Martins, Luciana Moreira
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 13/12/2011 PT
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O sistema de expressão de proteínas que utiliza baculovírus como vetor tem sido intensamente utilizado para a produção de proteínas recombinantes, pois ele permite a formação de modificações pós-traducionais e conta com um forte promotor, a poliedrina. Porém, em alguns casos, esse sistema não é tão eficaz, pois a infecção das células pelo vírus induz morte por apoptose, limitando a produção industrial de várias proteínas recombinantes de interesse. Reportamos a ocorrência de apoptose em cultivos de células de insetos e de mamíferos com depleção de nutrientes ou induzidos por agentes químicos e virais, e a suplementação dessas culturas com hemolinfa de Lonomia obliqua é capaz de estender a viabilidade da cultura evitando a morte por apoptose. Como objetivo, verificamos previamente o mecanismo de ação antiapoptótica de um isolado protéico da hemolinfa de L. obliqua. A identificação deste mecanismo poderá auxiliar no desenvolvimento de estratégias para controlar a apoptose nos cultivos permitindo a otimização da produtividade viral e de proteínas recombinantes.; The protein expression system using baculovirus as a vector has been intensively used for the production of recombinant proteins because it allows the formation of post-translational modifications and has a strong promoter...

Aspectos bioquímicos da hemolinfa e do casulo coletivo de Rhynchosciara americana; Biochemical aspects of hemolymph and cocoon collective Rhynchosciara Americana

Terra, Walter Ribeiro
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 14/06/1972 PT
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Os resultados obtidos nesta tese podem ser distribuídos em três grupos: composição química da hemolinfa e do casulo coletivo e determinação química de alguns componentes principais do corpo gorduroso e túbulos de Malpighi ao longo do desenvolvimento. Os principais resultados referentes à química da hemolinfa de larvas maduras são: 1) A hemolinfa possui uma densidade 1,043, pH = 7,27, osmolaridade = 216 miliosmoles e corresponde a 37% do pêso-úmido do animal e 26% de seu pêso-sêco. A hemolinfa não se coagula e possui um volume de células correspondente a 0,3% de seu volume total. 2) A análise química realizada deu conta de 88% do peso-sêco total da hemolinfa e revelou que entre os componentes presentes mais importantes em massa estão as proteínas, seguidas dos aminoácidos livres, enquanto que os osmóticamente mais ativos são os aminoácidos livres seguido de Mg++ e Na+. Entre os aminoácidos é notável a presença de ornitina e cistationina em concentrações relativamente elevadas e a ausência, mesmo em traços, de cisteína e/ou cistina e citrulina. 3) Os peptídeos ocorrem em concentrações elevadas, mas em pequeno número, e são compostos de 2 a 3 resíduos de aminoácidos em média; o mais abundante dos quais deve ser um dipeptídeo de histidina e ácido aspártico. 4) Citrato é o ânion mais importante da hemolinfa...

Nitric oxide production in blowfly hemolymph after yeast inoculation

Faraldo, A. C.; Sa-Nunes, A.; Del Bel, E. A.; Faccioli, L. H.; Lello, E.
Fonte: Elsevier B.V. Publicador: Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 240-246
ENG
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Although insects lack the adaptive immune response of the mammalians, they manifest effective innate immune responses that include both cellular and humoral components. Cellular responses are mediated by hemocytes and Immoral responses include the activation of proteolytic cascades that initiate many events, including NO production. In this work, we determined NO production in Chrysomya megaccphala hemolymph and hemocytes after yeast inoculation. Assays were performed with non-infected controls (NIL), saline-injected larvae (SIL) or larvae injected with Saccharomyces cerevisiae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24 or 48 h post-injection. NO levels in SIL were comparable to those measured in NIL until 12 h, which might be considered the basal production, increasing at 24 and 48 h post-injection, probably in response to the increased larval fragility after cuticle rupture. YIL exhibited significantly higher levels of NO than were found in other groups, peaking at 24 h. L-NAME and EDTA caused a significant reduction of NO production in YIL at this time, suggesting the activity of a Ca2+ -dependent NOS. Plasmatocytes and granular cells phagocytosed the yeasts. Plasmatocytes initiated the nodule formation and granular cells were the only hemocyte type to produce NO. These results permit us to conclude that yeasts induced augmented NO production in C. megacephala hemolymph and granular cells are the hemocyte type involved with the generation of this molecule. (c) 2005 Elsevier B.V. All rights reserved.

Morphological and quantitative aspects of nodule formation in hemolymph of the blowfly Chrysomya megacephala (Fabricius, 1794)

Faraldo, A. C.; Gregorio, E. A.; Lello, E.
Fonte: Academic Press Inc. Elsevier B.V. Publicador: Academic Press Inc. Elsevier B.V.
Tipo: Artigo de Revista Científica Formato: 372-377
ENG
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Insects manifest effective immune responses that include both cellular and humoral components. Morphological and quantitative aspects of cellular and Immoral cooperation during nodule formation in Chrysomya megacephala hemolymph against Saccharomyces cerevisae yeast cells were demonstrated for the first time. The analyses were performed in non-injected larvae (NIL), saline-injected larvae (SIL) and yeast-injected larvae (YIL). The hemolymph of injected groups was collected 0.5, 1, 2, 4, 12, 24, 36, or 48-h post-injection. Morphological aspects of YIL nodulation were investigated using transmission electron microscopy (TEM). Quantitative analyses consisted of total (THC) and differential hemocyte counts (DHC) in all the groups and total yeast count (TYC) in YIL, which were performed in an improved Neubauer chamber. Nodule formation was initiated at approximately 2-h post-injection. Twelve hours after the injection, TEM revealed the presence of an amorphous membrane, at the same time that circulating hemocyte number decreased significantly contrasting the increase of yeast number. Our results showed the ability of C megacephala hemolymph to perform humoral encapsulation when hemocyte population is insufficient to eliminate the microorganisms...

Metabolic Post-feeding Changes in Fat Body and Hemolymph of Dipetalogaster maximus (Hemiptera:Reduviidae)

Canavoso,Lilián E; Rubiolo,Edilberto R
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/1998 EN
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Lipids and glycogen in fat body as well as the modifications in the wet weight of this organ were evaluated in an unfed insect, Dipetalogaster maximus, on day 5 after adult ecdysis (time 0) and during a 30-day period after ingestion of blood meal. Total lipids, high density lipophorin (HDLp), carbohydrates, total proteins and uric acid were determined in the hemolymph during the same period. Fat body wet weight was maximum on day 10 post-feeding and represented on day 30 only 42% of the maximum weight. Lipids stored in the fat body increased up to day 15 reaching 24% of the total weight of tissue. Glycogen was maximum on day 20, representing approximately 3% of the fat body weight. HDLp represented at all times between 17-24% of the total proteins, whose levels ranged between 35 and 47 mg/ml. Uric acid showed at 20, 25 and 30 days similar levels and significantly higher than the ones shown at days 10 and 15. Hemolymphatic lipids fluctuated during starvation between 3-4.4 mg/ml and carbohydrates showed a maximum on day 15 after a blood meal, decreasing up to 0.26 mg/ml on day 25. The above results suggest that during physiological events such as starvation, the availability of nutrients is affected, involving principally the fat body reserves

Worker honeybee hemolymph lipid composition and synodic lunar cycle periodicities

Fonte: Associação Brasileira de Divulgação Científica Publicador: Associação Brasileira de Divulgação Científica
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/02/1997 EN
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The aim of the present investigation was to extend a previous study, showing a correlation of the variations of hemolymph carbohydrates with synodic lunar-like cycle and its circaseptan harmonics to worker honeybee hemolymph lipids. Hemolymph lipid concentrations of emerging worker imagos were analyzed in terms of one ideal synodic lunar cycle and processed by the cosinor method testing the null hypothesis versus the presence of 29.5-, 14.8- or 7.4-day periods in the data. A rhythmicity statistically compatible with a 29.5-day rhythm was observed for triacylglycerols and steroids as well as for body weight. A circadiseptan rhythm was determined for 1,3 diacylglycerols, while fatty acids and phospholipids exhibited a circaseptan rhythm. An agreement of peaks for triacylglycerols, steroids and body weight at the new moon, but not at the full moon, was noted with respect to trehalose and glucose circadiseptan rhythms. The latter moon-phase timing of peaks and nadirs, compared with that previously determined for trehalose and glucose, appeared to be identical to the circadiseptan rhythm and reciprocal for the circaseptan rhythms of 1,3 diacylglycerols. Reciprocal tendencies in circaseptans of trehalose and glucose on the one hand, and fatty acids and phospholipids on the other are indicated. The underlying causal nexus of these relationships is unknown

Performance evaluation of CHO-K1 cell in culture medium supplemented with hemolymph

Raffoul,Tássia; Swiech,Kamilla; Arantes,Mabel Karina; Sousa,Álvaro Paiva Braga de; Mendonça,Ronaldo Zucatelli; Pereira,Carlos Augusto; Suazo,Cláudio Alberto Torres
Fonte: Instituto de Tecnologia do Paraná - Tecpar Publicador: Instituto de Tecnologia do Paraná - Tecpar
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2005 EN
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The aim of this work was to evaluate the potential of hemolymph utilization as a culture medium supplement to cultivate the animal cell CHO-K1. For this purpose 1% v/v of hemolymph was added to DMEM medium containing 10% v/v of FBS and 1 or 4.5 g/L of glucose. The culture was grown in spinner flasks incubated in a 10% v/v CO2 environment, at 37ºC, with the Cytodex 1 microcarrier. Comparing the results obtained from the culture with hemolymph against those without hemolymph, a positive influence of the hemolymph was observed, as the experiment with hemolymph presented a 52% higher cell concentration and a higher productivity of up to 40%.

Characterization of adenosine deaminase (ADA) in Hemolymph of Biomphalaria glabrata

Vale,M. R.; Pereira,R. V.; Almeida,S. M.; Almeida,Y. M.; Nunes,S. F. L. C.
Fonte: Instituto Internacional de Ecologia Publicador: Instituto Internacional de Ecologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/05/2005 EN
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Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 µmols.L-1. Highest activity was found at 37ºC. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 µL of pure hemolymph per assay, pH 6.8, and 37ºC. This enzyme showed activities of 834 ± 67 µmol.min-1.L-1 (25ºC) and 2029 ± 74 µmol.min-1.L-1 (37ºC), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43ºC or 70% at 50ºC for 15 minutes. The ADA lost from 26 to 78% of its activity when hemolymph was pre-incubated at 50ºC for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions...

Ovary histology and quantification of hemolymph proteins of Rhipicephalus (Boophilus) microplus treated with Melia azedarach

Sousa,Lorena Alessandra Dias de; Rocha,Thiago Lopes; Saboia-Morais,Simone Maria Teixeira; Borges,Ligia Miranda Ferreira
Fonte: Colégio Brasileiro de Parasitologia Veterinária Publicador: Colégio Brasileiro de Parasitologia Veterinária
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2013 EN
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This study aimed to analyze ovary histology and quantify total protein in the hemolymph of Rhipicephalus (Boophilus) microplus females treated with hexane extracts from green fruits of Melia azedarach. Eight engorged females were immersed in the extract at 0.25% concentration, and eight in water containing 5% acetone (control). The females were dissected 72 hours after treatment, and the ovaries were weighed and subjected to standard histological techniques. The total protein concentration was measured in the hemolymph of 200 females, of which 100 were treated as described above and 100 served as a control. In the treated group, ovary weight reduction and predominance of immature oocytes were observed. In addition, there were decreases in the diameters of the cytoplasm and germ vesicle of the oocytes in the treated group, compared with the controls. The protein concentration in the hemolymph was higher in the treated group than in the controls. The morphological changes observed in the treated ovaries included: presence of vacuolization; alteration of oocyte morphology, which changed from rounded to elongated; deformation of the chorion; and disorganization of the yolk granules. These results demonstrate the action of M. azedarach fruit extracts on R. (B.) microplus oogenesis.

Indigenous Bacteria in Hemolymph and Tissues of Marine Bivalves at Low Temperatures

Olafsen, Jan A.; Mikkelsen, Helene V.; Giæver, Hanne M.; Høvik Hansen, Geir
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1993 EN
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Hemolymph and soft tissues of Pacific oysters (Crassostrea gigas) kept in sand-filtered seawater at temperatures between 1 and 8°C were normally found to contain bacteria, with viable counts (CFU) in hemolymph in the range 1.4 × 102 to 5.6 × 102 bacteria per ml. Pseudomonas, Alteromonas, Vibrio, and Aeromonas organisms dominated, with a smaller variety of morphologically different unidentified strains. Hemolymph and soft tissues of horse mussels (Modiolus modiolus), locally collected from a 6- to 10-m depth in the sea at temperatures between 4 and 6°C, also contained bacteria. The CFU in horse mussel hemolymph was of the same magnitude as that in oysters (mean, 2.6 × 104), and the bacterial flora was dominated by Pseudomonas (61.3%), Vibrio (27.0%), and Aeromonas (11.7%) organisms. In soft tissues of horse mussels, a mean CFU of 2.9 × 104 bacteria per g was found, with Vibrio (38.5%), Pseudomonas (33.0%), and Aeromonas (28.5%) constituting the major genera. After the challenge of oysters in seawater at 4°C to the psychrotrophic fish pathogen Vibrio salmonicida (strains NCIMB 2245 from Scotland and TEO 84001 from Norway) and a commensal Aeromonas sp. isolated from oysters, the viable count in hemolymph increased 1,000-fold to about 105 bacteria per ml. In soft tissues...

Bacterial Flora of the Hemolymph of the Blue Crab, Callinectes sapidus: Most Probable Numbers

Tubiash, Haskell S.; Sizemore, Ronald K.; Colwell, Rita R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/1975 EN
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The hemolymph of 290 freshly collected blue crabs from Chincoteague Bay, Va., was sampled over a 15-month period from August 1968 through November 1969 and most probable numbers of bacteria were determined by tube dilution. The hemolymph of 18% of all crabs sampled was found to be sterile, with 16% sterility in summer and 23% in winter samples. Despite individual variations, male crabs as a group had a higher bacterial hemolymph burden than females, and among both sexes summer counts were higher than winter. The hemolymph of crabs with missing appendages had significantly higher counts than uninjured crabs. The annual mean hemolymph most probable numbers per ml was 2,756 for males, 1,300 for females, and 1,876 for both sexes. The higher bacterial levels found in the hemolymph of male crabs may, in part, be explained by the fact that males, which predominated in the summer samples, had a higher incidence of injury and missing appendages than did females.

Cations in Hemolymph and Alimentary Tract Tissues of Healthy and Milky Diseased European Chafer (Amphimallon majalis) Larvae1

Steinkraus, K. H.; Field, C. C.; Kochansky, M. C.; Kaegebein, M. E.; Tashiro, H.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1973 EN
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A study was made of certain cations present in hemolymph and alimentary tract tissues of healthy and diseased European chafer larvae and the spores of Bacillus popilliae collected from diseased hemolymph. The major ions found in the hemolymph, in order of decreasing abundance, were potassium, magnesium, sodium, and calcium. Hemolymph of diseased larvae contained relatively higher concentrations of sodium, potassium, magnesium, iron, and zinc than hemolymph of healthy larvae. Concomitantly, the concentrations of ions were lower in the mid-gut and anterior intestinal tissues of diseased larvae. Only sodium decreased slightly in the diseased tissues of the rectum and rectal sac; other ions remained unchanged or increased. Little or no manganese or copper was detected in the hemolymph or tissues. The major cations of spores of B. popilliae were sodium, calcium, and magnesium. Small amounts of potassium, manganese, copper, iron, and zinc were detected in the spores. Based on calcium and dipicolinic acid determinations of the spores, sufficient calcium was found to allow for the formation of calcium dipicolinate in the expected concentrations.

Interactions between Sex-Transformation Mutants of DROSOPHILA MELANOGASTER. I. Hemolymph Vitellogenins and Gonad Morphology

Ota, T.; Fukunaga, A.; Kawabe, M.; Oishi, K.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1981 EN
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27.45%
In Drosophila, vitellogenins (yolk protein precursors) are synthesized by the female fat body, secreted into the hemolymph and subsequently taken up by the developing oocytes. The male fat body, on the other hand, does not do this even when immature ovaries are transplanted into the body cavity and grow. Thus, the hemolymph vitellogenins serve as an easily detectable sexually dimorphic biochemical marker.——We have examined hemolymph vitellogenins by SDS polyacrylamide gel electrophoresis in flies carrying various sex-transformation mutants (dsx, tra, tra-2 and tra-2OTF) singly and in all possible combinations. Chromosomal females homozygous for tra or tra-2 have no detectable hemolymph vitellogenins, while those homozygous for tra-2OTF exhibit appreciable levels of these proteins. Flies homozygous for dsx, both X/X and X/Y, have hemolymph vitellogenins, although the amount is consistently smaller in the latter. Indeed, X/Y; dsx/dsx is the only genotype in which hemolymph vitellogenins are detected in the X/Y flies. A clear hierarchy of epistasis exists among these sex-transformation mutants when they are examined in various combinations: dsx > tra, tra-2 > tra-2OTF. Moreover, an interaction between tra-2OTF and tra was seen in these experiments: X/X; tra-2OTF/tra-2OTF flies show the presence of only a trace of hemolymph vitellogenins when they are made heterozygous for tra. These results...

Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade*

Gorman, Maureen J.; Wang, Yang; Jiang, Haobo; Kanost, Michael R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Melanization, an insect immune response, requires a set of hemolymph proteins including pathogen recognition proteins that initiate the response, a cascade of mostly unknown serine proteinases, and phenoloxidase. Until now, only initial and final proteinases in the pathways have been conclusively identified. Four such proteinases have been purified from the larval hemolymph of Manduca sexta: hemolymph proteinase 14 (HP14), which autoactivates in the presence of microbial surface components, and three prophenoloxidase-activating proteinases (PAP1-3). In this study, we have used two complementary approaches to identify a serine proteinase that activates proPAP3. Partial purification from hemolymph of an activator of proPAP3 resulted in an active fraction with two abundant polypeptides of ∼32 and ∼37 kDa. Labeling of these polypeptides with a serine proteinase inhibitor, diisopropyl fluorophosphate, indicated that they were active serine proteinases. N-terminal sequencing revealed that both were cleaved forms of the previously identified hemolymph serine proteinase, HP21. Surprisingly, cleavage of proHP21 had occurred not at the predicted activation site but more N-terminal to it. In vitro reactions carried out with purified HP14 (which activates proHP21)...

Hemolymph Amino Acid Analysis of Individual Drosophila Larvae

Piyankarage, Sujeewa C.; Augustin, Hrvoje; Grosjean, Yael; Featherstone, David E.; Shippy, Scott A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50–300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations...

Measurement of Neuropeptides in Crustacean Hemolymph via MALDI Mass Spectrometry

Chen, Ruibing; Ma, Mingming; Hui, Limei; Zhang, Jiang; Li, Lingjun
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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Neuropeptides are often released into circulatory fluid (hemolymph) to act as circulating hormones and regulate many physiological processes. However, the detection of these low-level peptide hormones in circulation is often complicated by high salt interference and rapid degradation of proteins and peptides in crude hemolymph extracts. In this study, we systematically evaluated three different neuropeptide extraction protocols and developed a simple and effective hemolymph preparation method suitable for MALDI MS profiling of neuropeptides by combining acid-induced abundant protein precipitation/depletion, ultrafiltration, and C18 micro-column desalting. In hemolymph samples collected from crab Cancer borealis several secreted neuropeptides have been detected, including members from at least five neuropeptide families, such as RFamide, allatostatin, orcokinin, tachykinin-related peptide (TRP), and crustacean cardioactive peptide (CCAP). Furthermore, two TRPs were detected in the hemolymph collected from food-deprived animals, suggesting the potential role of these neuropeptides in feeding regulation. In addition, a novel peptide with a Lys-Phe-amide C-terminus was identified and de novo sequenced directly from the Cancer borealis hemolymph sample. To better characterize the hemolymph peptidome...

Insect hemolymph clotting : evidence for interaction between the coagulation system and the prophenoloxidase activating cascade

Li, Dongmei; Scherfer, Christoph; Korayem, Ahmed M.; Zhao, Zeng Qi; Schmidt, Otto; Theopold, Ulrich J.
Fonte: Pergamon-Elsevier Publicador: Pergamon-Elsevier
Tipo: Artigo de Revista Científica
Publicado em //2002 EN
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Here we describe a novel approach to isolate proteins involved in insect hemolymph coagulation. In order to avoid problems in purifying clot proteins after they had been crosslinked, we performed an in vitro coagulation reaction with cell-free hemolymph from the lepidopteran Galleria mellonella and used the resulting complexes to produce a specific antiserum. The antiserum reacted with a subset of hemolymph proteins as well as with granular cells, but not with other hemocyte types of Galleria. Screening expression libraries identified some positive clones, which turned out to code for some previously characterized components of immune cascades, as well as some novel candidates for clotting factors. Known components include members of both the coagulation system and the prophenol-activating cascade, lending support to the idea that both systems work together during the formation of a hemolymph clot. Novel candidates for insect clotting factors include a mucin-like protein, a glutathione-S-transferase, and a distant member of the α-crystallin/small heat shock protein family. Using assays measuring the activity of transglutaminase, a key enzyme in clotting reactions in both vertebrates and invertebrates, we found a partial overlap between transglutaminase substrates and proteins recognized by the antiserum against the in vitro-induced clot.; http://www.elsevier.com/wps/find/journaldescription.cws_home/390/description#description; D. Li...

Antibacterial activity in the hemolymph of the catarina scallop Argopecten ventricosus

Luna-González,Antonio; Maeda-Martínez,Alfonso; Campa-Córdova,Ángel; Orduña-Rojas,Javier
Fonte: UAM, Unidad Iztapalapa, Publicador: UAM, Unidad Iztapalapa,
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2007 EN
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We conducted a search for antibacterial peptide like activity in hemolymph of Argopecten ventricosus. Pre-purification of peptides was done by reverse phase HPLC. Hemolymph acidic supernatant was loaded into a column packed with C18 matrix. Stepwise elutions were performed with 5, 50, 80, and 100 % acetonitrile (ACN) in 0.05 % trifluoroacetic acid (TFA) over 550 min at a flow rate of 0.2 ml/min. Absorbance was monitored at 280 nm. Eluted fractions were concentrated under vacuum. Vibrio alginolyticus 138-2 was used as a model to test fractions. Growth inhibition zone were observed at 5 and 50 % ACN. This is the first report of antimicrobial peptide-like activity in hemolymph of a pectinid species.